The first three enzymatic steps in the de novo synthesis of pyrimidines (carbamyl phosphate synthetase II, aspartate transcarbamylase, and dihydroorotase) are the same in all cells, whether prokaryotic or eukaryotic. The structure, organization, and regulation of these three enzymatic activities and their respective genes vary among species. In bacteria, each enzyme activity is associated with a separate protein. In mammals, they are found together in a single translation product of 240,000 daltons called CAD. Our long term goals are to learn how having these three enzymes in one protein benefit mammals, how the de novo synthesis of pyrimidines contributes to normal development and cellular proliferation, and what new agents can be developed to block this pathway in cancer cells. By placing hamster CAD CDNA, whole or pieces, into expression vectors and placing these vectors in bacterial or hamster call mutants defective in one or more of these enzymes, we have developed a powerful system for studying the structure, function, and regulation of a multifunctional protein that plays a key role in an important biosynthetic pathway. A major focus of this proposal is to use directed mutagenesis to alter the hamster CAD protein, and, thus gain insight into its structure and relationship to cellular metabolism. To achieve this a combination of genetic, molecular biological and biochemical approaches will be utilized.